Can Age-Related Changes in Bone-Specific Alkaline Phosphatase in
Mares and Foals Be Detected With an Assay Developed for Humans? 1
K. J. Bowen, C. M. Hill, T. A. Poland, J. M. Kouba and J.E. Minton
Department of Animal Sciences and Industry
Kansas State University
ABSTRACT
A commercially available enzyme-linked immunosorbant assay for human bone specific alkaline
phosphatase was used to test the hypothesis that the assay could detect age-related
differences in concentrations of the enzyme in plasma from mares and their neonatal foals.
Jugular vein plasma was obtained from mares at parturition and from their neonatal foals prior
to suckling. The plasma was stored frozen until used for the assay. As expected, concentrations
of immunoreactive bone specific alkaline phosphatase were greater (P < 0.0001) in foals (747.0
± 33.2 μg/L) than mares (29.7 ± 33.2 μg/L). The assay was then subjected to two further
validation steps. To demonstrate quantitative recovery of added mass, human bone specific
alkaline phosphatase was added to samples of equine plasma. The concentration in the spiked
plasma was determined in the assay. The ratio of the concentration measured to the expected
concentration was 92.4 %, demonstrating acceptable recovery of added mass performance.
However, the second validation criteria, linearity of dilution could not be demonstrated. When
equine plasma was diluted 1.25- and 2.5-fold, the ratio of the concentration determined in the
assay to that expected was 128.0 % and 218.1 %, respectively. We conclude that the assay was
capable of detecting immunoreactive alkaline phosphatase in horses. This was evident by the
expected difference between mares and their neonatal foals. However, additional validation
and optimization steps will be required before this assay will be acceptable for providing
accurate estimates of circulating bone specific alkaline phosphatase data from horses.
1
Submitted in partial fulfillment of the requirements of GENAG 515 and the College of Agriculture Honors
program. The authors gratefully acknowledge the donation of assay reagents from Dr. Susan K. Durham, Technical
Manager, Immunodiagnostic Systems, Inc.
1
Introduction
Equine racing and showing enterprises in the United States encourage owners and trainers to
produce young horses that achieve mature body size at a young age. This means that
structurally immature horses must gain weight rapidly. Unfortunately, this practice often
results in undesirable leg and joint soundness anomalies (Kronfeld et al., 1990). There is
considerable interest in alternative management approaches that balance rapid growth with
life-long leg and joint soundness. This interest has spawned a search for reliable markers of
bone turnover in young horses that might be predictive of desirable bone growth. Bone specific
alkaline phosphatase represents one of those markers (Pagani et al., 2005).
Alkaline phosphatase is a ubiquitous enzyme found in many tissues and is mainly associated
with membranes and cell surfaces of small intestine, kidney, bone, liver and placenta (Pagani et
al., 2005). A single gene encodes tissue aspecific alkaline phosphatase and the protein product
possesses identical amino acid sequence for the liver, bone and kidney isoenzymes (Moss,
1986). The tissue aspecific forms of alkaline phosphatase are subjected to variable degrees of
post-translational processing (primarily varying degrees of glycosylation) resulting in tissuespecific isoforms for liver, bone and kidney (Moss, 1982).
Following post-translational processing, considerable amounts of the tissue associated isoforms
can be detected in the peripheral circulation. In healthy adults, the liver and bone-specific
isoforms circulate in a ratio of about 1:1 (Pagani et al., 2005). In horses, this enzyme has been
localized to growth plate cartilage. Its precise role in bone calcification is not understood, but a
commonly held view is that it is a marker of nascent bone (Henson et al., 1995). The prominent
role of this enzyme in the process of bone formation has lead to its emergence as an attractive
peripheral serum marker of bone turnover (Pagani et al., 2005). Consistent with its role in bone
mineralization, circulating bone-specific alkaline phosphatase is elevated considerably in very
young foals (Hank et al., 1993; Price et al., 2001), remains high though the first 6 to 8 mo of age,
then gradually diminishes to lower basal concentrations as horses advance in age beyond 1 yr
(Price et al., 2001; Jackson et al., 2003).
An analytical challenge to widespread use of bone-specific alkaline phosphatase in research and
clinical applications of horses is availability of a rapid, accurate, and technically streamlined
method for analysis in peripheral circulation. Wheat germ lectin selectively precipitates bone
specific alkaline phosphatase, and this characteristic has been exploited to develop a two-step
assay for the bone isoenzyme (Behr and Barnert, 1986). This assay has been adapted for use in
horses (Hank et al., 1993). Unfortunately, this assay measures the enzyme indirectly, is subject
to variation across batches of wheat germ (Behr and Barnert, 1986), and lacks the
attractiveness of a rapid and quantitative “off the shelf” assay kit.
2
The objective of the current study was to evaluate a commercially-available enzyme-linked
immunosorbant assay (ELISA) for human bone specific alkaline phosphatase for use in horses.
Although the amino acid sequence of equine bone specific alkaline phosphatase is yet to be
determined, we were encouraged by a report of a human immunoradiometric assay detecting
immunoreactive bone specific alkaline phosphatase in horse serum (Jackson et al., 1996).
Moreover, when the Basic Local Alignment Search Tool (BLAST) BLASTX function was run using
a human bone/liver/kidney alkaline phosphatase sequence (accession NM_000478; this
function takes a nucleotide sequence and compares it to translated proteins in the National
Center for Biotechnology Information (NCBI) database), the human protein sequence had
greater than 90% homology with cat, mouse, dog, rat, and cattle sequences
(http://www.ncbi.nlm.nih.gov/BLAST/ accessed April 26, 2007). Therefore, our hypothesis was
that the commercial ELISA could detect differences in neonatal foal and mare serum. A related
activity was to subject the assay to routine validation steps including recovery of added mass
and linearity of dilution.
Materials and Methods
The current study was conducted using plasma samples collected from a thesis research project
conducted at Kansas State University (Poland, 2006). In brief, sixteen Quarter Horse-type mares
(age 4 to 19 years) and their neonatal foals (< 1 d) were used. Jugular blood was collected after
parturition, but prior to foal suckling. Samples were collected into vacuum tubes containing
sodium heparin, and plasma was recovered following centrifugation and stored at -18 C until
analysis.
Bone-specific alkaline phosphatase was determined using an ELISA (OSTASE® Bone Specific
Alkaline Phosphatase EIA; product code AC-20F1 from Immunodiagnostic Systems Inc). The
package insert from the manufacturer provided evidence (from human clinical samples) that
the assay was specific for the bone iosenzyme. To further validate the assay for use in equine
serum, quantitative recovery of added mass and linearity of equine plasma dilution were
determined. Quantitative recovery was determined by adding known amounts of human bone
specific alkaline phosphatase standard from the OSTASE kit to equine plasma samples prior to
their analysis in the assay. To determine linearity of dilution, equine samples were evaluated
undiluted (neat), then diluted 1.25 and 2.5-fold, and evaluated in the assay. The dynamic range
of the standard curve was 5 (sensitivity of the assay) to 85.8 μg/L. All samples were analyzed in
a single assay run that had an intra-assay CV of 11.3%.
Mare and foal plasma bone specific alkaline phosphatase data were analyzed using the Mixed
Procedure of the Statistical Analysis System (SAS). The model included only age effects (mares
vs foals). Data represent the mean ± SEM of 16 horse per age group.
3
Results and Discussion
P < 0.0001
800
Bone-Specific Alkaline
Phosphatase,  g/L
As expected, immunoreactive bone specific
600
alkaline phosphatase was substantially greater
in neonatal foal plasma than in plasma from
400
their dams (Figure 1; P < 0.0001).
200
Concentrations estimated in individual mares
ranged from 8.6 to 67.0 μg/L, whereas foals
0
Mares
Foals
ranged from 360.8 to 1044.7 μg/L. Our results
Age
agree favorably with age associated changes in
horses reported previously using the wheat
Figure 1. Immunoreactive bone specific
alkaline phosphatase in mares and their
germ lectin binding assay (Hank et al., 1993;
neonatal foals. Each bar represents the mean
Price et al., 2001). However, it is not possible to
± SEM of 16 horses in each age group.
compare the concentrations measured in the
current assay to those determined in the wheat
germ lectin assay as the latter values are always expressed in units of activity per volume of
serum.
Horse 1
Horse 2
Horse 3
Horse 4
BAP Measured, g/L
200
150
100
50
0
0
50
100
150
200
hBAP Added to Equine Plasma, g/L
Figure 2. Recovery of added human (h) bone
specific alkaline phosphatase (BAP) in the
OSTASE enzyme-linked immunosorbant
assay when added to equine plasma.
The results for the test of quantitative recovery
are illustrated in Figure 2. When increasing
amounts of human bone specific alkaline
phosphatase were added to equine plasma, the
ratio of the measured concentration to the
expected concentration averaged 92.4 %. This
level of recovery is very typical of
immunoassays for hormones, growth factors
and cytokines published previously from our
laboratory (Minton and Cash, 1990; Griffith and
Minton, 1992; Minton and Parsons, 1993; Balaji
et al., 2000; Jenkins et al., 2004; Musser et al.,
2004).
4
Ratio of Measured to
Expected Concentration of
BAP x 100
Unfortunately, we could not demonstrate
linearity of dilution for the assay. Linearity of
300
dilution was tested in multiple runs, varying
200
incubation times and utilizing an array of equine
plasma samples. But, in every case, the assay
100
consistently over-reported the concentration
based upon the concentration in the undiluted
0
Neat
1.25
2.5
sample. Sample results at 1, 1.25, and 2.5-fold
Dilution of Equine Plasma
dilutions are illustrated in Figure 3 where
samples diluted 1.25-fold measured 128.0 % and
Figure 3. Ratio of bone specific alkaline
those diluted 2.5-fold measured 218.1 % of the
phosphatase (BAP) measured in the OSTASE
expected concentration. Because the foal
ELISA in undiluted (neat) plasma and plasma
samples averaged close to 800 μg/L and the
diluted 1.25- and 2.5 fold. Symbols represent
standard curve of the assay only extended to
individual data points and horizontal lines
represent the mean ratio at each dilution.
85.8 μg/L, it would be essential to dilute these
samples to obtain an accurate estimation of their
concentration. We do not know what factor(s) in the equine plasma account for this matrix
effect. One candidate may be the heparin used as an anticoagulant in the study. If this is the
case, it is possible that use of serum instead of plasma might be an easy solution to the
problem. Another solution may involve the addition of a plate reagent designed to be added to
all wells prior to addition of the standards and samples. According to the package insert, the
carrier for the standards contains a bovine protein matrix (most likely bovine serum albumin)
and only 50 μL of standard or sample are added to each well. There remains plenty of depth in
the well to add a buffer solution containing 4 to 5 % equine serum albumin to provide an
equine serum-like matrix. However, these solutions to the linearity of dilution issue will have to
be tested, and may not solve the problem.
In conclusion, although the OSTASE ELISA failed to fully validate for use in equine plasma, the
assay found immunoreactive alkaline phosphatase as evidence by the expected and
unmistakable concentration difference between mares and their neonatal foals. Although our
results are promising, additional validation and optimization steps will be required before this
assay will be acceptable for providing bone specific alkaline phosphatase data from horses to
be published in respected peer-reviewed scientific journals.
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